Bacteria play important roles in human health. Bacterial communities are important components of normal physiology, as revealed by studies of human microbiota. In contrast, pathogenic bacteria cause morbidity and mortality. Whether impacting health or disease, interactions of bacteria with hosts share many common features. To survive and thrive, bacteria must monitor their intracellular and extracellular environments and elicit appropriate adaptive responses to changing conditions. Two-component system? (TCS) phosphotransfer pathways involving a sensor histidine protein kinase and a phosphorylation-activated response regulator that generates the output response comprise a versatile regulatory scheme that occurs in hundreds of thousands of regulatory systems. While structure and function of core elements are conserved, TCSs display enormous diversity. Data acquired from numerous studies of individual systems as well as global analyses have revealed differences in the magnitudes of enzyme activities, affinities of macromolecular interactions, levels of signaling proteins and system architecture, all of which presumably contribute to tuning response behavior to the needs of individual systems. The overarching goal of this research is to understand design principles of TCSs to the extent that system behavior can be predicted, or at least rationalized, with knowledge of system parameters. Protein concentrations are known to be critical parameters that influence reaction kinetics and outcomes in vitro, yet they are commonly overlooked in cellular studies. Histidine kinase and response regulator concentrations and stoichiometry are known to differ greatly among TCSs, but the effects of these variations on system behavior, other than robustness, are largely unstudied. This project will fill this gap by exploring how histidine kinase and response regulator concentrations impact system design and behavior. Investigations will be performed using a set 19 Escherichia coli TCSs. Approaches will utilize reporter gene assays and measurement of intracellular phosphorylation of response regulators to quantitate response output, mass spectrometry to quantitate levels of two-component proteins in cells under un-induced and activated conditions, mathematical modeling with experimental data to determine kinetics parameters and predict system behavior, and competition assays in continuous cultures to assess fitness. Studies will address four broad questions. Does the size of a TCS regulon place a requirement on the level of response regulator in a TCS? How does the stoichiometry of histidine kinases and response regulators impact response output in an activated TCS? How are system parameters configured to accommodate differences in histidine kinase and response regulator concentrations? Does non-specific phosphorylation of response regulators place a requirement on the phosphatase activity of histidine kinases? These investigations will identify core design principles of TCSs and how variations in individual parameters are accommodated in different systems. Principles uncovered in this study of TCSs are likely to be broadly applicable to other regulatory systems.